www.nature.com/scientificreports OPEN Short term changes in the proteome of human cerebral organoids induced by 5-MeO-DMT Received: 5 May 2017 Vanja Dakic1,2, Juliana Minardi Nascimento 1,3, Rafaela Costa Sartore1,2, Renata de Moraes Accepted: 14 September 2017 Maciel1, Draulio B. de Araujo4, Sidarta Ribeiro4, Daniel Martins-de-Souza 3,5 & Stevens K. Published: xx xx xxxx Rehen 1,2 Dimethyltryptamines are entheogenic serotonin-like molecules present in traditional Amerindian medicine recently associated with cognitive gains, antidepressant effects, and changes in brain areas related to attention. Legal restrictions and the lack of adequate experimental models have limited the understanding of how such substances impact human brain metabolism. Here we used shotgun mass spectrometry to explore proteomic differences induced by 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) on human cerebral organoids. Out of the 6,728 identified proteins, 934 were found differentially expressed in 5-MeO-DMT-treated cerebral organoids.In silico analysis reinforced previously reported anti-inflammatory actions of 5-MeO-DMT and revealed modulatory effects on proteins associated with long-term potentiation, the formation of dendritic spines, including those involved in cellular protrusion formation, microtubule dynamics, and cytoskeletal reorganization. Our data offer the first insight about molecular alterations caused by 5-MeO-DMT in human cerebral organoids. Dimethyltryptamines are naturally-occurring molecules hypothesized to be involved in spontaneous altered states of consciousness such as dreams, free imagination, and insightful creativity1,2. N,N-dimethyltryptamine (N,N-DMT) and bufotenine (5-HO-DMT) have been traditionally used as entheogens by Amerindians3,4 as major active ingredients of Virola snuff and a brew called Ayahuasca5. The popularity of Ayahuasca as part of religious ceremonies continues to spread in South America and other countries6, possibly motivated by its strong antidepressant effects7,8. Chronic Ayahuasca ingestion has been associated with cognitive gains and structural brain changes in areas related to attention, self-referential thought, and internal mentation9,10. Another member of this group of molecules is 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), secreted in large amounts by Incilius alvarius11,12. 5-MeO-DMT has been used more recently by the Seris, an indigenous group from the state of Sonora, in Mexico. The search for the molecular mechanisms underlying the effects of dimethyltryptamines showed that N,N-DMT and 5-MeO-DMT, two closely related metabolic products, can act as systemic endogenous regulators of inflammation and immune homeostasis through both 5-hydroxytryptamine receptors (5-HTRs) and sigma-1 receptors (σ-1Rs)13,14. Under severe hypoxia, N,N-DMT robustly increased the survival of in vitro cultured human cortical neurons, monocyte-derived macrophages, and dendritic cells acting through σ-1Rs15. The direct evidence of neuroimmune communication and neuroregenerative effects of N,N-DMT and 5-MeO-DMT greatly enhanced expectations for psychedelic research. Our limited understanding of the physiological activity of dimethyltryptamines and other classic psyche- delic substances is caused not only by legal restrictions on such research16,17 but also by the lack of adequate experimental models18–20. In the past few years, considerable progress has been made regarding the neural differentiation of human pluripotent stem cells into mature neurons and cerebral organoids21. Human neural 1D’Or Institute for Research and Education (IDOR), Rio de Janeiro, Brazil. 2Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil. 3Laboratory of Neuroproteomics, Institute of Biology, Department of Biochemistry and Tissue Biology, University of Campinas (UNICAMP), Campinas, Brazil. 4Brain Institute, Federal University of Rio Grande do Norte, Natal, Brazil. 5Instituto Nacional de Biomarcadores em Neuropsiquiatria (INBION), Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Sao Paulo, Brazil. Vanja Dakic and Juliana Minardi Nascimento contributed equally to this work. Correspondence and requests for materials should be addressed to S.K.R. (email: [email protected]) SCIENTIFIC REPORTS | 7: 12863 | DOI:10.1038/s41598-017-12779-5 1 www.nature.com/scientificreports/ Figure 1. Effects of 5-MeO-DMT on hNPCs. (A) Expression of mRNA for internal control (GAPDH), SR1, 5-HT2A, and 5-HT2C in hNPCs. (B) Confirmation of σ-1R protein (green) expression by immunocytochemistry, phalloidin showing the cytoskeleton (red) and DAPI staining nuclei (blue), scale bar 20 μm. (C) Quantification of cell proliferation based on EdU staining after treatment with 5-MeO-DMT. (D) Percentage of dead cells in hNPCs treated with 5-MeO-DMT. (E–H) Effects of 5-MeO-DMT on neuronal arborization by quantification of (E) total neurite length (sum of the length of all neurites attached to the cell), (F) number of segments, (G) number of extremities, and (H) number of nodes type 1. Bar represents median. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test, and only p-values < 0.05 were considered significant. Here, all comparisons showed p-values > 0.05. progenitor cells (hNPC) are useful cell systems for high-throughput screening due to their homogeneity, along with little complexity and limited differentiation potential. On the other hand, cerebral organoids are complex, three-dimensional (3D) culture systems composed of multiple cell types that self-organize into various brain regions similarly to those in vivo, including the cerebral cortex, ventral forebrain, midbrain–hindbrain boundary, and hippocampus22,23. Combining different cell types in a complex 3D configuration can better simulate brain biology and function. As such, cerebral organoids can reproduce the function and architecture of the brain, especially regarding development and neuronal plasticity. A comparison of gene expression programs of human fetal neocortex and in vitro cortical development by single-cell RNA sequencing found remarkable similarities24. Cerebral organoids may well recapitulate environmental effects on human nervous system, particularly related to plasticity and growth24–26, and circumvent problems of discrepancies in metabolic pathways occurring in transla- tional studies involving animal models. The development of such a model offers an exciting new range of oppor- tunities to investigate the molecular responses of human neural tissue to psychoactive substances. Here we analyzed the effect of 5-MeO-DMT on human neural cells and cerebral organoids. By employing mass spectrometry-based proteomics to analyze cerebral organoids, we managed to investigate effects on a large scale and in an unbiased manner, and also gained insight into its molecular mechanisms and biochemical path- ways27. To the best of our knowledge, our results are the first to show that 5-MeO-DMT modulates proteins involved in long-term potentiation (LTP), in addition to morphogenesis and maturation of dendritic spines, while inhibiting neurodegeneration and cell death. Results Human neural progenitor cells are unaffected by 5-MeO-DMT. First, we examined the effects of 5-MeO-DMT on hNPCs (detailed characterization in28). hNPCs showed basal expression of σ-1Rs but not 5-HT2A or 5-HT2C receptors (Fig. 1A,B). Using a high-content screening analysis, we tested the effects of 5-MeO- DMT (23 nM to 7.11 μM) on hNPC death, proliferation, and differentiation. There was no evidence of change in cell death or proliferation in response to 5-MeO-DMT (Fig. 1C,D). In addition, by quantifying some aspects of dendritic branch complexity, we measured neural arborization based on MAP2 staining of young neurons exposed to 5-MeO-DMT compared to an unexposed control. Despite a slight trend, there were no statistically significant differences in the measured parameters (Fig. 1E–H, all p > 0.05). Human cerebral organoids express 5-MeO-DMT receptors. The lack of alterations in cell death, pro- liferation, and differentiation/arborization in hNPCs exposed to 5-MeO-DMT could be due to the low cellular diversity and lack of complex interactions between different cell types. Thusly, we tested human cerebral orga- noids, which better replicate the complexity and function of in vivo neural circuitry. In 45 day-old cerebral orga- noids, basal immunostaining was observed in AMPA (selective glutamate receptor 2) and NMDA (ionotropic glutamate receptor) – both characteristic of glutamatergic synapses – along with the neuronal marker MAP2 SCIENTIFIC REPORTS | 7: 12863 | DOI:10.1038/s41598-017-12779-5 2 www.nature.com/scientificreports/ Figure 2. Cerebral organoids express 5-MeO-DMT receptors and different cell type markers. (A) Cerebral organoids presenting smooth texture and homogeneous coloring at 45 days of differentiation (scale bar 1000 μm). (B) Cerebral organoids are composed of several cell types, including mature neurons, as shown by MAP2 staining. (C) Cells expressing AMPAR1 are found at the organoid edge, while (D) cells expressing NMDAR1 and (E) GFAP are detected within the organoid. (F) Cells positive for 5-HT2A receptor, and (G) σ-1R, the primary molecular targets for 5-MeO-DMT, are also found in the organoid. Scale bars: A = 1000 μm; B = 50 μm; C, D, E, F, and G = 20 μm. (H) The expression of molecular targets for 5-MeO-DMT was also confirmed by RT- PCR. (Fig. 2A–D), as previously described29. Glial cells (GFAP+) are also present in organoids, as shown in Fig.